ENaC Subunits Research Articles

Impact of Genetic Variation of αENaC on Clinical Presentation in Cystic Fibrosis

ObjectiveIndividuals with the same disease causing variant of cystic fibrosis (CF) can have very different clinical outcomes, suggesting that yet‐to‐be identified modifier genes may play an important role in clinical presentation and disease progression. The epithelial sodium channel (ENaC) plays an important role in the pathophysiology of CF. Previous research has shown that substitution of alanine for threonine at position 663 of the SCNNA1 gene, which encodes the alpha subunit of ENaC, results in channel gain‐of‐function. In this study we sought to examine how genetic variation in the SCNNA1 gene influences clinical outcomes in CF.MethodsBuccal swabs were collected from thirty‐five CF patients that had at least one copy of the del.F508 mutation (19 males, 16 females). Samples were analyzed for allelic status at position 663 of SCNNA1 (AA, AT/TT). Medical charts were reviewed for resting vitals, pulmonary function, and indicators of disease progression.ResultsTwenty‐one subjects were homozygous for the alanine allele (AA, A663), 12 were heterozygous (AT) and 2 were homozygous for the threonine allele (TT). Due to low numbers in the TT group the AT and TT groups were combined for analysis (T663). Individuals in the T663 group had significantly higher resting heart rate (88±9 v 77±9, p=0.004) and tended to have lower body weight (133.7±56.5lbs v 143.6±31.9lbs), and higher systolic blood pressure (121±16.9mmHg v 115±16.5mmHg) compared to the A663 group. Forced vital capacity was significantly lower in the T663 group (3.64±1.12L v 4.29±0.94L, p=0.038). However FEV1/FVC was higher in the T663 group (75.0±12.1% v 71.8±11.0%, p=0.043), suggesting smaller lung volumes but not greater obstructive lung disease compared to the A663 group. There was no statistically significant difference in the frequency of pancreatic insufficiency or cystic fibrosis related type‐I diabetes between the two groups.ConclusionWe found that CF patients with the T663 allele tended to have smaller lungs as well as higher resting heart rate and blood pressure when compared to the A663 group. This finding suggests that the T663 variant results in cardiovascular compensation for altered pulmonary function.Support or Funding InformationNoneThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Salt Loading Effects on ENaC and Blood Pressure in Aged 129Sv Mice

Essential hypertension in the elderly population is a major public health problem because these patients usually do not respond well to antihypertensive drugs. More than half the cases of essential hypertension are thought to be associated with salt sensitivity from excessive dietary salt intake. We hypothesized that salt loading augments renal epithelial sodium channel expression and blood pressure in aged 129Sv mice. The renal epithelial sodium channel regulates total body salt balance and blood pressure, but mechanisms for its regulation by dietary salt intake in the aged kidney are not completely known. Twelve month old 129Sv mice were subject to salt loading for 4 weeks. Water intake, urine production, blood pressure, and electrolytes in the urine were measured before and after salt loading. Both the total and cleaved forms of ENaC subunits were assessed by Western blotting and densitometric analysis. ENaC alpha protein was augmented in the kidneys of salt‐loaded hypertensive 129Sv mice compared to non‐salt loaded mice. Water intake, urine output, and systolic blood pressure significantly increased in the 129Sv mice after salt‐loading. Salt loading resulted in an increase in urinary sodium levels and a decrease in urinary potassium levels. These findings may provide new links between salt‐sensitivity, ENaC expression, and blood pressure regulation in the aged kidney.Support or Funding InformationThis project was funded by the U.S. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (Grant K01 DK099617; to A.A.A.) and the University of Florida College of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mapping the sites of localization of epithelial sodium channel (ENaC) and CFTR in segments of the mammalian epididymis.

The sperm produced in the seminiferous tubules pass through the rete testis, efferent ducts, and epididymis. The epididymis has three distinct regions known as caput, corpus, and cauda. The transit through the epididymis is an essential process in sperm maturation. The lumen of each epididymal region has a unique fluid composition regulated by many ion channels and transporters in the epithelial cells. The objective of this study was to map the sites of localization of ion channels ENaC and CFTR along the length of the mouse and rat epididymis using confocal microscopic imaging. The integrity of the fine structure of the tissues was verified by fluorescent phalloidin staining of actin filaments visualized by high-resolution confocal microscopy. The 2D and 3D images showed preservation of the stereocilia. Based on these images we determined morphometric parameters of the epithelial cells and ducts. ENaC and CFTR immunofluorescence appeared almost continuously on the apical membrane of caput and in smooth muscle myoid cells. In cauda, CFTR expression was observed continuously in long stretches of epithelium interrupted by clusters of cells that showed no CFTR expression. Similar patterns of localization were observed in both mouse and rat samples. Mutations in the CFTR gene are known to result in male infertility. Based on the widespread presence of ENaC along the epididymis we suggest that mutations in ENaC subunits may also be associated with male infertility. The diverse phenotypes associated with CFTR mutations may be due to malfunction of CFTR at specific subcellular locations in the male reproductive system.

Proliferative regulation of alveolar epithelial type 2 progenitor cells by human Scnn1d gene.

Lung epithelial sodium channel (ENaC) encoded by Scnn1 genes is essential for maintaining transepithelial salt and fluid homeostasis in the airway and the lung. Compared to α, β, and γ subunits, the role of respiratory δ-ENaC has not been studied in vivo due to the lack of animal models.Methods: We characterized full-length human δ802-ENaC expressed in both Xenopus oocytes and humanized transgenic mice. AT2 proliferation and differentiation in 3D organoids were analysed with FACS and a confocal microscope. Both two-electrode voltage clamp and Ussing chamber systems were applied to digitize δ802-ENaC channel activity. Immunoblotting was utilized to analyse δ802-ENaC protein. Transcripts of individual ENaC subunits in human lung tissues were quantitated with qPCR.Results: The results indicate that δ802-ENaC functions as an amiloride-inhibitable Na+ channel. Inhibitory peptide α-13 distinguishes δ802- from α-type ENaC channels. Modified proteolysis of γ-ENaC by plasmin and aprotinin did not alter the inhibition of amiloride and α-13 peptide. Expression of δ802-ENaC at the apical membrane of respiratory epithelium was detected with biophysical features similar to those of heterologously expressed channels in oocytes. δ802-ENaC regulated alveologenesis through facilitating the proliferation of alveolar type 2 epithelial cells.Conclusion: The humanized mouse line conditionally expressing human δ802-ENaC is a novel model for studying the expression and function of this protein in vivo .

Pre-eclampsia is associated with altered expression of the renal sodium transporters NKCC2, NCC and ENaC in urinary extracellular vesicles.

Pre-eclampsia is a hypertensive disorder of pregnancy characterised by hypertension and sodium retention by the kidneys. To identify changes in sodium uptake proteins in the tubules of the distal nephron, we studied their expression in urinary extracellular vesicles or exosomes (uEVs). Urine was collected from women with pre-eclampsia or during normal pregnancy, and from healthy non-pregnant controls. uEVs were isolated by centrifugation and analyzed by Western blot. Expression, proteolytic cleavage and phosphorylation was determined by densitometric analysis normalized to the exosome marker CD9. Results showed a significant increase in phosphorylation of the activating S130 site in NKCC2, the drug target for frusemide, in women with pre-eclampsia compared with normal pregnant women. Phosphorylation of the activating sites T101/105 in NKCC2 was similar but the activating T60 site in NCC, the drug target for thiazide diuretics, showed significantly less phosphorylation in pre-eclampsia compared with normal pregnancy. Expression of the larger forms of the α subunit of ENaC, the drug target for amiloride, was significantly greater in pre-eclampsia, with more fragmentation of theγ subunit. The differences observed are predicted to increase the activity of NKCC2 and ENaC while reducing that of NCC. This will increase sodium reabsorption, and so contribute to hypertension in pre-eclampsia.

Epithelial sodium channel biogenesis and quality control in the early secretory pathway.

The epithelial sodium channel, ENaC, is responsible for Na reabsorption in several epithelia and is composed of homologous α, β, and γ subunits. Here, we will explore the differential regulation of ENaC subunits during biogenesis in the early secretory pathway. ENaC subunits are subject to numerous posttranslational modifications, including glycosylation, protease activation, disulfide bond formation, palmitoylation, and glycosylation, each of which modulate channel function. For example, glycan addition is regulated by sodium and affects protease activation at the cell surface, protein trafficking, sodium-dependent regulation, and sodium transport. Glycosylation of the α subunit also determines whether a chaperone, Lhs1/GRP170, selects the protein for endoplasmic reticulum-associated degradation. Recognition by this chaperone is blocked by assembly of the ENaC transmembrane domains. In contrast, cytosolic lysines are acetylated in the early secretory pathway, which inhibits ubiquitination and endocytosis at the cell surface. As sodium reabsorption by ENaC in the distal nephron regulates salt and water homeostasis, ENaC function is critical for human health. Therefore, identifying and characterizing modifiers of ENaC in the early secretory pathway may provide both new therapeutic targets and further our basic understanding of membrane protein assembly and regulation.

ENaC in Cholinergic Brush Cells.

Cholinergic polymodal chemosensory cells in the mammalian urethra (urethral brush cells = UBC) functionally express the canonical bitter and umami taste transduction signaling cascade. Here, we aimed to determine whether UBC are functionally equipped for the perception of salt through ENaC (epithelial sodium channel). Cholinergic UBC were isolated from ChAT-eGFP reporter mice (ChAT = choline acetyltransferase). RT-PCR showed mRNA expression of ENaC subunits Scnn1a, Scnn1b, and Scnn1g in urethral epithelium and isolated UBC. Scnn1a could also be detected by next generation sequencing in 4/6 (66%) single UBC, two of them also expressed the bitter receptor Tas2R108. Strong expression of Scnn1a was seen in some urothelial umbrella cells and in 65% of UBC (30/46 cells) in a Scnn1a reporter mouse strain. Intracellular [Ca2+] was recorded in isolated UBC stimulated with the bitter substance denatonium benzoate (25 mM), ATP (0.5 mM) and NaCl (50 mM, on top of 145 mM Na+ and 153 mM Cl− baseline in buffer); mannitol (150 mM) served as osmolarity control. NaCl, but not mannitol, evoked an increase in intracellular [Ca2+] in 70% of the tested UBC. The NaCl-induced effect was blocked by the ENaC inhibitor amiloride (IC50 = 0.47 μM). When responses to both NaCl and denatonium were tested, all three possible positive response patterns occurred in a balanced distribution: 42% NaCl only, 33% denatonium only, 25% to both stimuli. A similar reaction pattern was observed with ATP and NaCl as test stimuli. About 22% of the UBC reacted to all three stimuli. Thus, NaCl evokes calcium responses in several UBC, likely involving an amiloride-sensitive channel containing α-ENaC. This feature does not define a new subpopulation of UBC, but rather emphasizes their polymodal character. The actual function of α-ENaC in cholinergic UBC—salt perception, homeostatic ion transport, mechanoreception—remains to be determined.

Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice

BackgroundThe epithelial sodium channel ENaC consists of three subunits encoded by Scnn1a, Scnn1b, and Scnn1g and increased sodium absorption through this channel is hypothesized to lead to mucus dehydration and accumulation in cystic fibrosis (CF) patients. MethodsWe identified potent and specific antisense oligonucleotides (ASOs) targeting mRNAs encoding the ENaC subunits and evaluated these ASOs in mouse models of CF-like lung disease. ResultsASOs designed to target mRNAs encoding each ENaC subunit or a control ASO were administered directly into the lungs of mice. The reductions in ENaC subunits correlated well with a reduction in amiloride sensitive channel conductance. In addition, levels of mucus markers Gob5, AGR2, Muc5ac, and Muc5b, periodic acid-Schiff's reagent (PAS) goblet cell staining, and neutrophil recruitment were reduced and lung function was improved when levels of any of the ENaC subunits were decreased. ConclusionsDelivery of ASOs targeting mRNAs encoding each of the three ENaC subunits directly into the lung improved disease phenotypes in a mouse model of CF-like lung disease. These findings suggest that targeting ENaC subunits could be an effective approach for the treatment of CF.

Tamoxifen Decreases Lithium-Induced Natriuresis in Rats With Nephrogenic Diabetes Insipidus.

Lithium is widely used in the treatment of bipolar affective disorders, but often causes nephrogenic diabetes insipidus (NDI), a condition characterized by a severe urinary concentrating defect. Lithium-induced NDI is associated with dysregulation of the amiloride-sensitive epithelial sodium channel (ENaC), which is essential for renal sodium reabsorption. Sex hormones have been shown to affect the expression of aquaporin-2 (AQP2) and sodium transporters. Therefore, we evaluated whether tamoxifen (TAM), a selective estrogen receptor modulator (SERM), would affect lithium-induced dysregulation of ENaC subunits and natriuresis. Rats were fed with lithium-containing food for 2 weeks to induce NDI and natriuresis. TAM was administered daily via gastric gavage after 1 week of lithium administration. Lithium treatment alone resulted in increased urinary sodium excretion and significant reduction of βENaC and γENaC at both RNA and protein levels. In addition, the plasma sodium level reduced after lithium treatment. Administration of TAM prevented increased urinary sodium excretion as well as attenuated the downregulation of βENaC and γENaC. Consistent with these findings, immunohistochemistry (IHC) showed stronger labeling of βENaC and γENaC subunits in the apical domain of the collecting duct cells in the cortical tissue of lithium-fed rats treated with TAM. Other major sodium transporters including NaPi-2, NKCC2, Na/K-ATPase, and NHE3, are believed not to have an effect on the increased urinary sodium excretion since their expression increased or was unchanged after treatment with lithium. In conclusion, the results demonstrated that TAM rescued the adverse effects of the lithium-induced increase in fractional excretion of sodium after the establishment of lithium-induced NDI.

Role of the Scaffold Protein MIM in the Actin-Dependent Regulation of Epithelial Sodium Channels (ENaC)

Epithelial Sodium Channels (ENaCs) are expressed in different organs and tissues, particularly in the cortical collecting duct (CCD) in the kidney, where they fine tune sodium reabsorption. Dynamic rearrangements of the cytoskeleton are one of the common mechanisms of ENaC activity regulation. In our previous studies, we showed that the actin-binding proteins cortactin and Arp2/3 complex are involved in the cytoskeleton-dependent regulation of ENaC and that their cooperative work decreases a channel's probability of remaining open; however, the specific mechanism of interaction between actin-binding proteins and ENaC is unclear. In this study, we propose a new component for the protein machinery involved in the regulation of ENaC, the missing-in-metastasis (MIM) protein. The MIM protein contains an IMD domain (for interaction with PIP2 -rich plasma membrane regions and Rac GTPases; this domain also possesses F-actin bundling activity), a PRD domain (for interaction with cortactin), and a WH2 domain (interaction with G-actin). The patch-clamp electrophysiological technique in whole-cell configuration was used to test the involvement of MIM in the actin-dependent regulation of ENaC. Co-transfection of ENaC subunits with the wild-type MIM protein (or its mutant forms) caused a significant reduction in ENaC-mediated integral ion currents. The analysis of the F-actin structure after the transfection of MIM plasmids showed the important role played by the domains PRD and WH2 of the MIM protein in cytoskeletal rearrangements. These results suggest that the MIM protein may be a part of the complex of actin-binding proteins which is responsible for the actin-dependent regulation of ENaC in the CCD.

Incorporation of the δ-subunit into the epithelial sodium channel (ENaC) generates protease-resistant ENaCs in Xenopus laevis

The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits (α, β, and γ), but a fourth δ-subunit may replace α and form atypical δβγ-ENaCs. Using Xenopus laevis as a model, here we found that mRNAs of the α- and δ-subunits are differentially expressed in different tissues and that δ-ENaC predominantly is present in the urogenital tract. Using whole-cell and single-channel electrophysiology of oocytes expressing Xenopus αβγ- or δβγ-ENaC, we demonstrate that the presence of the δ-subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the α- and γ-subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike α-ENaC, the δ-subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by δβγ-ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the δ-subunit prevented cleavage of γ-ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the Xenopus δ-ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution.

Molecular genetic studies in a case series of isolated hypoaldosteronism due to biosynthesis defects or aldosterone resistance.

Hypoaldosteronism is associated with either insufficient aldosterone production or aldosterone resistance (pseudohypoaldosteronism). Patients with aldosterone defects typically present with similar symptoms and findings, which include failure to thrive, vomiting, hyponatremia, hyperkalemia and metabolic acidosis. Accurate diagnosis of these clinical conditions therefore can be challenging. Molecular genetic analyses can help to greatly clarify this complexity. The aim of this study was to obtain an overview of the clinical and genetic characteristics of patients with aldosterone defects due to biosynthesis defects or aldosterone resistance. We investigated the clinical and molecular genetic features of 8 consecutive patients with a clinical picture of aldosterone defects seen in our clinics during the period of May 2015 through October 2017. We screened CYP11B2 for aldosterone synthesis defects and NR3C2 and the three EnaC subunits (SCNN1A, SCNN1B and SCNN1G) for aldosterone resistance. We found 4 novel and 2 previously reported mutations in the genes CYP11B2, NR3C2, SCNN1A and SCNN1G in 9 affected individuals from 7 unrelated families. Molecular genetic investigations can help confidently diagnose these conditions and clarify the pathogenicity of aldosterone defects. This study may expand the clinical and genetic correlations of defects in aldosterone synthesis or resistance.

DISTINCT EFFECTS OF GLUTATHIONE DISULFIDE ON δ‐ AND α‐EPITHELIAL SODIUM CHANNEL SUBUNITS

BackgroundAmiloride‐sensitive epithelial sodium channel (ENaC) is a membrane bound ion channel with selective inward permeability to Na. ENaC plays a critically important role in maintaining net fluid homeostasis. To date four ENaC subunits (α‐, β‐, γ‐, and δ‐ENaC) have been cloned in mammalian species. Mice, however, only express αβγ. In humans, an additional ENaC subunit (δ1) and its slice variant (δ2) have been recently identified. Unlike αβγ‐ENaC, the physiological role and regulation of channels made up of δ1 and δ2 subunits remain unclear.HypothesisWe tested the hypothesis that sodium channels containing α and δ ENaC subunits (alongside β and γ) are differentially regulated under oxidative stress.MethodsUsing the Xenopus laevis oocyte expression system, full‐length cDNAs for human ENaC subunits, and two electrode voltage clamp measurements, we modeled antioxidant depletion (a form of oxidative stress) and compared the effects of oxidized glutathione (GSSG) on αβγ‐ENaC and δβγ‐ENaC whole cell current. High extracellular GSSG treatments (400μM) increases the redox potential, and models antioxidant depletion in an oocyte expression system. This experimental approach allows for the singular study of αβγ vs. δβγ ENaC in a cell model system capable of normal assembly of multimeric ion channels into the membrane.ResultsGSSG significantly decreased ENaC activity in oocytes expressing αβγ‐ENaC (n=10). Conversely, GSSG significantly increased whole cell current in oocytes expressing δ1βγ‐ENaC (n=12) and in δ2βγ‐ENaC expressing oocytes (n=13) immediately upon extracellular exposure. While both α and δ ENaC subunits are directly modified by GSSG at Cys thiol sites, the effect and mechanism of GSSG posttranslational modification of α vs δ expressing Na channels differed significantly in our studies. Heterologous expression of αβR566Xγ ENaC in oocytes indicate that GSSG proteolytically degrades classical αβγ ENaC. Whole cell recordings and biotinylation studies indicate that δ2 ENaC subunits increase net Na transport via insertion of new membrane protein, whereas GSSG plays no role in δ1 transport and insertion into the cell membrane.ConclusionWe found that αβγ ENaC activity is decreased by GSSG. Conversely δβγ ENaC activity is significantly increased by GSSG. This conclusion highlights the need to better understand the opposing translational effects of α vs δ ENaC subunits, as it may occur in human tissue. Additional studies are needed to better understand the molecular regulation and pathophysiological roles of α and δ ENaC subunits.Support or Funding InformationSupport for this project was made possible by the University of Utah Investigator Initiated Grant awarded to MNH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ACID SENSING ION CHANNEL‐2 LOCALIZES WITH α‐ AND γ‐, BUT NOT β‐ENAC SUBUNITS IN CILIATED AIRWAYS

The epithelial sodium channel (ENaC) and Acid‐sensing ion channels (ASIC) are members of a superfamily of channels that play a significant role in transepithelial cation transport. The protein structure of the subunits that form these channels share great similarity, with two transmembrane segments (TM1 and TM2) and a large extracellular component. ENaC subunits (α, β, γ and δ) have been identified in airways. However, the stoichiometry of the subunits that composed the ENaC channel remains elusive. ASIC2 expression has been reported in ciliated cells of trachea and nasal septum. Previous studies suggested that ENaC subunits and ASIC may form a functional hybrid of non‐selective cation channels (1–4). Herein, we hypothesized that ASIC2 is expressed in porcine airways and Na+ transport can be modulated by hybrid channels composed of ASIC2 and ENaC subunits. We obtained small pieces of peripheral porcine lung tissue of approximately 0.5 cm3 and pieces of trachea of approximately 0.5 cm2 that were dissected and immediately snap‐frozen at −80°C. Serial tissue sections (7 μm thick) containing cross‐sections of small airways (diameters 0.5 – 1 mm) were used for immunohistochemistry. We used polyclonal antibodies directed to ASIC2, α‐, β‐ and γ‐subunits of ENaC as well as monoclonal antibodies directed to β‐Tubulin and ZO1, the tight junction protein. Confocal microscopic analysis demonstrated that in distal airways ASIC2 and ENaC subunits were mainly localized in airway ciliated cells. ASIC2 was found distributed at the base of the multicilia, with a distribution similar to that observed for α‐ and γ‐ENaC subunits. In contrast, a strong signal for β‐ENaC subunit was observed along the cilia in a restricted group of airway ciliated cells. In proximal airways, ASIC2 signal was also identified at the base of the multicilia of ciliated cells, similar to the distribution of α‐ and γ‐ENaC subunits. Our data suggest that ASIC2 could combine with α‐ and γ‐ENaC subunits to form hybrids of non‐selective cation channels as previously indicated (1–4), and participate in mucociliary clearance, regulation of airway surface liquid volume and composition.Support or Funding InformationSupported by the Cystic Fibrosis Research Inc., and The Nancy Olmsted Trust for Pediatric Pulmonology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal Denervation Attenuates Nicotine‐Induced Increase in Thiazide‐Sensitive Na+/Cl− Cotransporter in the Young Pre‐Hypertensive Spontaneously Hypertensive Rat

Systemic infusion of nicotine induces renal inflammation and the development of premature hypertension in young spontaneously hypertensive rats (SHR). Renal denervation (RDN) prevents this nicotine-mediated hypertension and attenuates renal inflammation. We hypothesized that these findings were related to alterations in renal sodium handling. Following bilateral RDN or sham surgery in young (3–5 week old SHR), we continuously infused saline or nicotine bitartrate (15mg/kg/day) subcutaneously for 2 weeks. Urinary sodium was assessed via spectrophotometric analysis on spot urine samples obtained at the end of the infusion. Renal homogenates were assessed by Western blot for the presence of the epithelial sodium channel subunits (ENaC-alpha, -beta, and -gamma), sodium-potassium-chloride (NKCC2), and thiazide-sensitive sodium-chloride co-transporter (NCC). Systemic infusion of nicotine induced significant sodium retention, compared to saline, in Sham treated animals, as measured by a significant decrease in urinary sodium (from 252±55 mM to 137±26, p<0.05). During nicotine infusion in animals with RDN, there was a trend towards lesser sodium retention and a higher urinary sodium (192±46 mM, p=0.09). Western blot demonstrated that nicotine infusion in Sham treated animals induced a 1.7 fold increase in renal NCC expression, compared to saline (p<0.05). However, there was no nicotine effect on the expression of the three ENaC subunits or NKCC2 (p>0.05). RDN prevented the increase in renal NCC expression in response to nicotine infusion (p<0.05). We conclude that reduced renal sodium retention and decreased expression of NCC may contribute to the prevention of nicotine-mediated hypertension and renal inflammation by RDN. Support or Funding Information National Institutes of Health (NIH K08 HL119588, PPG HL14388) Spot urinary sodium following Sham surgery or RDN followed by 2 week infusion of subcutaneous saline or nicotine. Urine samples were collected at the end of infusion. Renal Thiazide-Sensitive Na+/Cl− Cotransporter (NCC) Expression Following Renal Denervation and 2-week Subcutaneous Infusion of Saline or Nicotine. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

N‐Glycan and Heparan Sulphate Components of the Extracellular Matrix are Essential for Laminar Shear Stress Response of the Epithelial Sodium Channel (ENaC)

The epithelial sodium channel (ENaC), a mechanosensitive ion channel, is known to be activated by laminar shear stress (LSS) [1]. We hypothesise that the large extracellular loops of ENaC interact with the complex extracellular matrix (ECM) and that the application of LSS deforms both the extracellular matrix (ECM) and the interacting ENaC components resulting in a change in ENaC activity. This study investigated whether or not the N‐glycan and heparan sulphate components of the ECM are specifically involved in the LSS activation of ENaC.Human α, β and γ ENaC subunits were co‐expressed in oocytes surgically harvested from female Xenopus laevis. Two‐electrode voltage‐clamp was used to determine ENaC activity in response to LSS when different components of the ECM were degraded by neuraminidase and heparinase III enzymes. A significant LSS response was observed in ENaC expressing oocytes, compared to native (no ENaC) oocytes; normalized LSS current of 5.5 ± 1.1 compared to 0.06 ± 0.03 (p = 0.003, n =8–14). In ENaC expressing oocytes cleavage of N‐glycan from the ECM with neuraminidase (0.2U/mL for 2h at 21°C) significantly reduced the normalised LSS response from 4.5 ± 0.4 to 1.7 ± 0.4 (p=0.003, n=8). Destruction of heparan sulphate components of the ECM with heparinase III (0.5 U/mL for 30min at 21°C) significantly reduced the LSS response from 5.1 ± 1.2 to 1.2 ± 0.5 (p=0.009, n=12). Data indicates that both removal of N‐glycan and cleavage of heparan sulphate from the ECM reduce the activation of ENaC by LSS, indicating that they are crucial components of the ECM for mechanosensation by ENaC.Support or Funding InformationThis study was supported by the Royal Society Marsden fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Subunit composition critically determines pH sensitivity of epithelial sodium channels in Xenopus laevis

The Epithelial Sodium Channel (ENaC) mediates the selective flux of sodium ions across the apical membrane of epithelial cells. ENaC consists of three subunits (α,β,γ) and its activity at the cell surface is subject to a multitude of regulatory mechanisms, including proteolytic cleavage, extracellular pH and Na+ ions. Channel regulation may also depend on ENaC subunit composition, as an additional δ‐subunit can replace α‐ENaC in the heterotrimeric configuration. As the physiological implications of ENaC containing the δ‐subunit are poorly understood, we characterized the regulation of Xenopus δβγ‐ENaC by extracellular pH and Na+.Regulation of Xenopus δβγ‐ENaC by extracellular Na+ and pH was investigated by two‐electrode voltage‐clamp and cell‐attached Patch‐Clamp electrophysiology using the Xenopus oocyte expression system. Successive reduction of extracellular pH from pH 8 – 6 increased amiloride‐sensitive transmembrane currents of oocytes expressing δβγ‐ENaC by &gt; 7 fold. In cell‐attached Patch‐Clamp recordings the pH of the pipette solution (pHpip = extracellular) affected δβγ‐ENaC open probability and kinetics. Compared to pHpip at 7.4, alkaline conditions (pHpip 8) decreased open probability as well as the mean channel open time, whereas under acidic conditions (pHpip 6) an increased open probability was accompanied by a reduction of average channel closed times. Transmembrane currents mediated by Xenopus δβγ‐ENaC are sensitive to sodium self‐inhibition, a phenomenon describing the fast inhibition of ENaC in the presence of high extracellular Na+ concentrations. Channel activation under acidic conditions (pH 7 &amp; 6) was accompanied by a decrease in magnitude and apparent Na+‐affinity of self‐inhibition, whereas these effects were reversed under alkaline conditions (pH 8). Hypothesizing mutual regulation of δβγ‐ENaC activity by extracellular Na+ and pH, we examined whether neutralization of pH‐sensitive aspartates in the δ‐subunit affected channel self‐inhibition and regulation by pH. Specific aspartates in a subunit domain corresponding to a putative Na+‐sensing site of mouse α‐ENaC (Kashlan et al., 2015, J Biol Chem 290:568–76) were replaced by asparagines via site‐directed mutagenesis (δD293N, δD296N, δD293,6N). All mutations decreased pH‐mediated channel activation. Notably, ENaC containing the δD296N mutation also displayed a decreased sodium self‐inhibition, which was not observed in channels bearing a single δD293N mutation. Furthermore, human δβγ‐ENaCs which have a reduced sodium self‐inhibition were not activated by acidic pH as observed with the Xenopus orthologue displaying a strong sodium self‐inhibition.In conclusion, these results demonstrate that activity of ENaC containing the δ‐subunit in Xenopus laevis is sensitive to changes in the extracellular pH, whereas ENaC containing the α‐subunit is not. Proton‐mediated activation of ENaC involves changes in single channel kinetics and a reduction of sodium self‐inhibition. Thus, sensitivity of this amphibian ENaC to extracellular pH is critically determined by its subunit composition.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Localization of epithelial sodium channel (ENaC) and CFTR in the germinal epithelium of the testis, Sertoli cells, and spermatozoa.

Spermatogenesis starts within the seminiferous tubules of the testis by mitotic division of spermatogonia that produces spermatocytes. Meiotic division of these spermatocytes produces haploid spermatids that differentiate into spermatozoa. In this study, we examined the expression of ENaC and CFTR (a Cl- channel) in rat testicular sections using confocal microscopic immunofluorescence. The structural integrity of the seminiferous tubule sections was verified by precise phalloidin staining of the actin fibers located abundantly at both basal and adluminal tight junctions. The acrosome forming regions in the round spermatids were stained using an FITC coupled lectin (wheat germ agglutinin). In all phases of the germ cells (spermatogonia, spermatocytes, and spermatids) ENaC was localized in cytoplasmic pools. Prior to spermiation, ENaC immunofluorescence appeared along the tails of the spermatids. In spermatozoa isolated from the epididymis, ENaC was localized at the acrosome and a central region of the sperm flagellum. The mature sperm are transcriptionally silent. Hence, we suggest that ENaC subunits in cytoplasmic pools in germ cells serve as the source of ENaC subunits located along the tail of spermatozoa. The locations of ENaC is compatible with a possible role in the acrosomal reaction and sperm mobility. In contrast to ENaC, CFTR immunofluorescence was most strongly observed specifically within the Sertoli cell nuclei. Based on the nuclear localization of CFTR we suggest that, in addition to its role as an ion channel, CFTR may have an independent role in gene regulation within the nuclei.

Oxymatrine attenuates lipopolysaccharide-induced acute lung injury by activating the epithelial sodium channel and suppressing the JNK signaling pathway.

The epithelial sodium channel (ENaC) and mitogen-activated protein kinase (MAPK) pathway have been reported to be associated with the progression of acute lung injury (ALI). Oxymatrine (OMT) alone or combined with other drugs can ameliorate paraquat- or oleic acid-induced lung injury. However, the effect of OMT on lipopolysaccharide (LPS)-induced ALI remains unknown. The aim of the present study was to evaluate whether OMT can attenuate LPS-induced ALI through regulation of the ENaC and MAPK pathway using an ALI mouse model. Histological assessment of the lung and inflammatory cell counts in bronchoalveolar lavage fluid (BALF) were performed by H&E and Wright-Giemsa staining. The lung wet/dry (W/D) weight ratio and the levels of tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), ENaC subunits, and the MAPK pathway members were determined. Isolated type II rat alveolar epithelial cells were incubated with OMT 30 min before LPS stimulation to investigate the activation of ENaC and the MAPK pathway. The results showed that OMT remarkably alleviated histopathologic changes in lung and pulmonary edema, reduced inflammatory cell counts in BALF, and decreased TNF-α and CRP levels in a dose-dependent manner. OMT significantly increased the three subunits of ENaC proteins in vivo and in vitro, while it decreased p-ERK/ERK, p-p38/p38, and p-JNK/JNK ratios in vivo. However, only the JNK pathway was markedly inhibited in vitro following pretreatment with OMT. Collectively, the results suggested that OMT might alleviate LPS-induced ALI by elevating ENaC proteins and inhibiting the JNK signaling pathway.

MnTBAP therapy attenuates the downregulation of sodium transporters in obstructive kidney disease.

Ureteral obstruction is associated with reduced expressions of renal sodium transporters, which contributes to impaired urinary concentrating capacity. In this study, we employed a synthetic mitochondrial superoxide dismutase 2 (SOD2) mimic MnTBAP to investigate the role of mitochondrial oxidative stress in modulating the sodium transporters in obstructive kidney disease. Following unilateral ureteral obstruction (UUO) for 7 days, a global reduction of sodium transporters including NHE3, NCC, NKCC2, and ENaCα was observed as determined by qRT-PCR, Western Blotting or immunohistochemistry. Among these sodium transporters, the downregulation of NHE3, NCC, and NKCC2 was partially reversed by MnTBAP treatment. In contrast, the reduction of ENaCα was not affected by MnTBAP. The β and γ subunits of ENaC were not significantly altered by ureteral obstruction or MnTBAP therapy. To further confirm the anti-oxidant effect of MnTBAP, we examined the levels of TBARs in the urine collected from the obstructed ureters of UUO mice and bladder of sham mice. As expected, the increment of urinary TBARs in UUO mice was entirely abolished by MnTBAP therapy, indicating an amelioration of oxidative stress. Meantime, we found that three types of SOD were all reduced in obstructed kidneys determined by qRT-PCR, which was unaffected by MnTBAP. Collectively, these results demonstrated an important role of mitochondrial oxidative stress in mediating the downregulation of sodium transporters in obstructive kidney disease.